The goal of the proposed studies is to develop high resolution 3-D Imaging Mass Spectrometry (IMS) technology to a level that it is capable of delivering high resolution images in a practical time frame. High spatial resolution IMS (~10 um) will be employed with tissue preparation protocols that provide 1 um matrix crystal sizes and high speed lasers (.3 kHz) to provide data on multiple serial sections in a efficacious and timely manner. It is planned to develop the protocols that would allow peptides, proteins, lipids and drugs and other small molecules to be tracked in a 3-D manner in a tissue volume. In addition, work is planned to accomplish the significant biocomputational effort needed to deal with data dimensionality and size reduction, normalization of ion intensities for the relative quantitation of images both within a single 2-D experiment and across serial sections of a tissue volume for 3-D reconstruction, registration of the sections with anatomic fidelity, and generation of a 3-D images of individual molecules in a practical time domain. Several important biological problems will be addressed with this technology; 1) assessment of the 3-D molecular distributions and boundaries of striosomes in the striatum of the mouse brain, substructures implicated in neuropsychiatric disorders such as Parkinsonism, Huntington's Disease, and obsessive-compulsive disorder, 2) 3-D mapping of molecular events in glioma growth, progression, and maintenance, both in the tumor and microenvironment, in the optic nerve of a mouse model with linkage to existing 3-D microscopy images in the brain atlas, 3) Co-registration of 3-D IMS data with other imaging modalities; studies of the disposition and metabolism of contrast agents used for PET imaging, the elucidation of molecular aspects in diffusion weighted imaging by MRI, and the co-registration/correlation of molecular events obtained by IMS with MR relaxation data. This work will bring together several powerful imaging modalities and, through the unique attributes of each, gain a combinatorial advantage in understanding whole tissue dynamics. In one aim, it is planned to combine microscopy and IHC measurements, MRI and IMS as part of a single 3-D volume that is easily interrogated using standard internet tools.